Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames study- Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be mutagenic under the conditions of this test only in S. typhimurium strain TA1535, this response was reproducible when utilizing the pre-incubation method.

Chromosome aberration- Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system.  The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Mouse Lymphoma AssayIn vitro- The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it was considered to be non-mutagenic in this assay.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 September 2017 Experimental completion date: 23 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Physical state/Appearance: Clear yellow liquid
Batch: 52611021
Purity: 100% (UVCB product)
Expiry Date: 12 January 2018
Storage Conditions: Room temperature in the dark
Target gene:
histidine locus in S. typhimurium and tryptophan locus in E. coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver S9 in standard co-factors
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, and 5000 µg/plate.
Vehicle / solvent:
The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Acetone was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
presence of S9-mix
Details on test system and experimental conditions:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were required for verification due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were performed due to spreading colonies to ensure an accurate count.

Test for Mutagenicity: Confirmatory Experiment – Pre-Incubation and Plate Incorporation Methods
As Experiment 2 was concluded to be positive in TA1535 only, a third, confirmatory experiment was performed using both the pre incubation and plate incorporation methods in the presence and absence of metabolic activation.
The dose range was determined by the results of Experiment 2 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.

Without Metabolic Activation
Performed as described in plate incorporation method.

With Metabolic Activation
Performed as described in plate incorporation method

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (acetone) control plates gave counts of revertant colonies generally within the normal range. Some of the vehicle/untreated control counts for TA1535 and TA98 in the first mutation test were just above the in-house control maxima. These counts were still considered acceptable as the majority of counts were within the historical profile and all were within the expected control range outlined for the strains in the GSP and the tester strains responded very well with the respective positive controls in both the presence and absence of S9 mix.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No toxicity was observed in the confirmatory experiment at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A greasy test item precipitate was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Small increases were noted in Experiment 1 (WP2uvrA at 150 and 5000 µg/plate in the absence of S9-mix), however these responses were within the in-house historical vehicle/untreated control values for the strain and were, therefore considered of no biological relevance.
In light of these results, Experiment 2 was conducted using the pre-incubation method.

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
Dose-related and statistically significant increases in TA1535 revertant colony frequency were observed at and above 500 µg/plate in the absence of S9-mix and from 150 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 1500 µg/plate with a maximum fold increase over the concurrent vehicle control of 9.3 at 5000 µg/plate. In the presence of S9, the mean colony counts were just in excess of the in-house upper historical control maxima from 150 µg/plate with a maximum fold increase over the concurrent vehicle control of 13.1 noted at 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the remaining Salmonella or Escherichia coli bacterial strains, with any dose of the test item, either with or without metabolic activation.

Experiment 3 (Confirmatory Test)
In light of the discrepancy in results between Experiments 1 and 2, a third test was conducted employing both the pre-incubation and plate incorporation methods and a single strain of Salmonella typhimurium, TA1535 in the absence and presence of S9-mix.
In the pre-incubation, statistically significant increases in TA1535 revertant colony frequency were observed, initially from 150 µg/plate in the absence of S9-mix and 50 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 1500 µg/plate with a maximum fold increase over the concurrent vehicle control of 6.5 at 5000 µg/plate. In the presence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 150 µg/plate with a maximum fold increase over the concurrent vehicle control of 12 noted at 5000 µg/plate.
In the plate incorporation assay, statistically significant increases in TA1535 revertant colony frequency were also observed initially from 50 µg/plate in the absence of S9-mix and 15 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 500 µg/plate with a maximum fold increase over the concurrent vehicle control of 11.9 at 5000 µg/plate. In the presence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 50 µg/plate with a maximum fold increase over the concurrent vehicle control of 21.1 noted at 5000 µg/plate.

Spontaneous Mutation Rates (ConcurrentNegativeControls)Experiment1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

95

 

30

 

22

 

49

 

18

 

120

(111)

38

(34)

29

(25)

51

(51)

21

(20)

119

 

35

 

23

 

52

 

22

 

 

Experiment 2

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

111

 

13

 

22

 

19

 

14

 

110

(114)

17

(15)

24

(26)

18

(19)

19

(17)

122

 

14

 

32

 

21

 

19

 

 

Experiment 3 – Confirmatory Test: Plate incorporation

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

TA1535

13

 

11

(12)

11

 

 

Experiment 3 – Confirmatory Test: Pre-Incubation

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

TA1535

12

 

17

(15)

15

 

Test Results: Experiment 1 – Without MetabolicActivation(Plate Incorporation)

 

Test Period

From: 06 October 2017

To: 09 October 2017

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

125

109

116

(117)

8.0#

32

30

34

(32)

2.0

31

26

19

(25)

6.0

47

43

45

(45)

2.0

14

12

11

(12)

1.5

 

1.5 µg

130

107

109

(115)

12.7

33

32

41

(35)

4.9

28

29

23

(27)

3.2

44

39

52

(45)

6.6

9

13

10

(11)

2.1

 

5 µg

112

119

120

(117)

4.4

29

27

31

(29)

2.0

24

23

24

(24)

0.6

50

46

48

(48)

2.0

11

11

15

(12)

2.3

 

15 µg

117

103

124

(115)

10.7

31

31

38

(33)

4.0

28

29

30

(29)

1.0

43

39

55

(46)

8.3

10

8

14

(11)

3.1

 

50 µg

137

133

126

(132)

5.6

36

34

36

(35)

1.2

22

27

20

(23)

3.6

49

41

40

(43)

4.9

12

13

11

(12)

1.0

 

150 µg

98

121

114

(111)

11.8

40

29

32

(34)

5.7

34

29

35

(33)

3.2

52

52

40

(48)

6.9

10

8

15

(11)

3.6

 

500 µg

93

134

116

(114)

20.6

33

32

30

(32)

1.5

29

26

31

(29)

2.5

38

44

44

(42)

3.5

11

7

9

(9)

2.0

 

1500 µg

80 P

104 P

85 P

(90)

12.7

29 P

35P

36P

(33)

3.8

35 P

33 P

26 P

(31)

4.7

49 P

51 P

47 P

(49)

2.0

10 P

13 P

12 P

(12)

1.5

 

5000 µg

111 P

98 P

103 P

(104)

6.6

33 P

35 P

31 P

(33)

2.0

35 P

31 P

33 P

(33)

2.0

45 P

51 P

49 P

(48)

3.1

11 P

11 P

8 P

(10)

1.7

Positive controls S9-Mix (-)

 

Name DoseLevel

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

598

608

524

(577)

45.9

923

1115

952

(997)

103.5

871

964

1029

(955)

79.4

178

214

216

(203)

21.4

235

393

309

(312)

79.1

 

 

ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

P           Test itemprecipitate

#            Standarddeviation


 

Test Results: Experiment 1 – With MetabolicActivation(Plate Incorporation)

 

Test Period

From: 06 October 2017

To: 09 October 2017

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

91

122

117

(110)

16.6#

38

36

39

(38)

1.5

40

34

38

(37)

3.1

51

53

50

(51)

1.5

17

15

22

(18)

3.6

 

1.5 µg

113

115

107

(112)

4.2

41

40

43

(41)

1.5

32

34

32

(33)

1.2

46

53

51

(50)

3.6

19

17

12

(16)

3.6

 

5 µg

102

123

115

(113)

10.6

39

44

38

(40)

3.2

41

45

45

(44)

2.3

50

57

55

(54)

3.6

16

15

19

(17)

2.1

 

15 µg

117

116

110

(114)

3.8

38

37

38

(38)

0.6

28

41

40

(36)

7.2

55

53

51

(53)

2.0

19

20

22

(20)

1.5

 

50 µg

104

119

98

(107)

10.8

42

43

40

(42)

1.5

34

36

34

(35)

1.2

48

58

52

(53)

5.0

21

20

16

(19)

2.6

 

150 µg

105

112

89

(102)

11.8

41

39

39

(40)

1.2

34

37

36

(36)

1.5

52

50

56

(53)

3.1

15

18

19

(17)

2.1

 

500 µg

112

106

101

(106)

5.5

43

45

39

(42)

3.1

33

26

30

(30)

3.5

52

49

57

(53)

4.0

18

19

20

(19)

1.0

 

1500 µg

99 P

101 P

105 P

(102)

3.1

42 P

46 P

41 P

(43)

2.6

35 P

36 P

30 P

(34)

3.2

51 P

51 P

54 P

(52)

1.7

22 P

17 P

19 P

(19)

2.5

 

5000 µg

105 P

110 P

122 P

(112)

8.7

47 P

37P

38P

(41)

5.5

39 P

41 P

40 P

(40)

1.0

55 P

56 P

50 P

(54)

3.2

22 P

24 P

17 P

(21)

3.6

Positive controls S9-Mix (+)

 

Name DoseLevel

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2368

2524

2509

(2467)

86.1

298

302

281

(294)

11.2

542

492

547

(527)

30.4

285

450

454

(396)

96.4

394

376

296

(355)

52.2

 

 

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

P      Test item precipitate

#            Standard deviation

Test Results: Experiment 2 – Without MetabolicActivation(Pre- Incubation)

 

Test Period

From: 13 October 2017

To: 16 October 2017

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

111

118

104

(111)

7.0#

11

11

21

(14)

5.8

29

26

23

(26)

3.0

15

25

15

(18)

5.8

18

18

18

(18)

0.0

 

15 µg

140

105

111

(119)

18.7

9

12

11

(11)

1.5

35

28

36

(33)

4.4

21

5

18

(15)

8.5

9

26

27

(21)

10.1

 

50 µg

88

99

99

(95)

6.4

14

10

14

(13)

2.3

30

23

23

(25)

4.0

21

23

19

(21)

2.0

28

19

21

(23)

4.7

 

150 µg

109

136

94

(113)

21.3

15

22

19

(19)

3.5

19

21

24

(21)

2.5

21

23

27

(24)

3.1

16

18

14

(16)

2.0

 

500 µg

103

124

121

(116)

11.4

25

35

25

** (28)

5.8

26

28

23

(26)

2.5

33

25

17

(25)

8.0

18

26

15

(20)

5.7

 

1500 µg

127 P

132 P

104 P

(121)

14.9

54 P

68 P

65 P

*** (62)

7.4

32 P

40 P

29 P

(34)

5.7

20 P

31 P

29 P

(27)

5.9

16 P

33 P

21 P

(23)

8.7

 

5000 µg

111 P

126 P

142 P

(126)

15.5

152 P

123 P

115 P

*** (130)

19.5

34 P

37 P

27 P

(33)

5.1

18 P

17 P

16 P

(17)

1.0

22 P

22 P

29 P

(24)

4.0

Positive controls S9-Mix (-)

 

Name DoseLevel

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1072

934

986

(997)

69.7

1278

1235

1592

(1368)

194.9

882

859

955

(899)

50.1

306

300

320

(309)

10.3

278

246

315

(280)

34.5

 

 

ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO      4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

P             Test item precipitate

**             p0.01

***           p0.00

#                Standard deviation


 

Test Results: Experiment 2 – With MetabolicActivation(Pre- Incubation)

 

Test Period

From: 13 October 2017

To: 16 October 2017

 

 

 

 

 

 

 

 

 

 

S9-Mix (+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

111

88

98

(99)

11.5#

9

20

12

(14)

5.7

40

32

30

(34)

5.3

30

30

24

(28)

3.5

20

21

14

(18)

3.8

 

15 µg

127

94

85

(102)

22.1

22

3

13

(13)

9.5

39

25

32

(32)

7.0

17

26

26

(23)

5.2

10

17

24

(17)

7.0

 

50 µg

93

106

110

(103)

8.9

28

34

22

(28)

6.0

36

31

44

(37)

6.6

34

24

22

(27)

6.4

17

24

19

(20)

3.6

 

150 µg

83

88

109

(93)

13.8

58

33

39

** (43)

13.1

34

40

34

(36)

3.5

13

27

34

(25)

10.7

13

10

15

(13)

2.5

 

500 µg

113

75

99

(96)

19.2

57

63

72

*** (64)

7.5

24

25

31

(27)

3.8

31

32

28

(30)

2.1

14

16

17

(16)

1.5

 

1500 µg

107 P

101 P

90 P

(99)

8.6

119 P

98 P

86 P

*** (101)

16.7

34 P

38 P

35 P

(36)

2.1

27 P

27 P

24 P

(26)

1.7

14 P

8 P

12 P

(11)

3.1

 

5000 µg

109 P

107 P

106 P

(107)

1.5

162 P

197 P

193 P

*** (184)

19.2

38 P

39 P

50 P

(42)

6.7

37 P

21 P

20 P

(26)

9.5

18 P

12 P

15 P

(15)

3.0

Positive controls S9-Mix (+)

 

Name DoseLevel

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2174

1392

2124

(1897)

437.8

282

276

299

(286)

11.9

228

328

323

(293)

56.3

213

233

251

(232)

19.0

511

497

526

(511)

14.5

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

P      Test itemprecipitate

**          p0.01

***        p0.001

#            Standarddeviation

Test Results: Confirmatory Experiment – With andWithoutMetabolic Activation(Pre-incubation)

 

 

Test Period

From: 19 October 2017

To: 22 October 2017

 

 

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strain

Without S9 TA1535

With S9 TA1535

Solvent Control (Acetone)

16

13

12

(14)

2.1#

12

10

12

(11)

1.2

 

15 µg

15

16

13

(15)

1.5

24

20

16

(20)

4.0

 

50 µg

12

25

23

(20)

7.0

43

24

38

*** (35)

9.8

 

150 µg

22

21

26

* (23)

2.6

51

56

80

*** (62)

15.5

 

500 µg

37

27

38

***  

(34)

6.1

80

69

67

*** (72)

7.0

 

1500 µg

64P

62P

73P

*** (66)

5.9

111P

94 P

95 P

*** (100)

9.5

 

5000 µg

99P

96P

77P

*** (91)

11.9

133P

133P

129P

*** (132)

2.3

 

Positive controls

 

Name DoseLevel

No. of Revertants

ENNG

2AA

5 µg

2 µg

417

501

561

(493)

72.3

321

315

347

(328)

17.0

 

ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine

2AA 2-Aminoanthracene

P                Test itemprecipitate

*                p 0.05

***           p0.001

#                Standard deviation


 

Test Results: Confirmatory Experiment – With andWithoutMetabolic Activation (PlateIncorporation)

 

Test Period

From: 19 October 2017

To: 22 October 2017

 

 

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strain

Without S9 TA1535

With S9 TA1535

Solvent Control (Acetone)

19

12

20

(17)

4.4#

12

9

23

(15)

7.4

 

15 µg

16

9

11

(12)

3.6

27

32

26

* (28)

3.2

 

50 µg

30

34

37

* (34)

3.5

84

58

71

*** (71)

13.0

 

150 µg

25

43

54

** (41)

14.6

120

103

108

*** (110)

8.7

 

500 µg

53

72

66

***(64)

9.7

173

156

155

*** (161)

10.1

 

1500 µg

113P

127P

107P

*** (116)

10.3

202P

180P

219P

*** (200)

19.6

 

5000 µg

198P

175P

237P

*** (203)

31.3

292P

345P

313P

*** (317)

26.7

 

Positive controls

 

Name DoseLevel

No. of Revertants

ENNG

2AA

5 µg

2 µg

345

383

420

(383)

37.5

191

146

173

(170)

22.6

                     


ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine2AA2-Aminoanthracene

P                Test itemprecipitate

*                p0.05

**             p0.01

***           p0.001

#                Standard deviation

Conclusions:
Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be mutagenic under the conditions of this test only in S. typhimurium strain TA1535, this response was reproducible when utilizing the pre-incubation method.
Executive summary:

 Introduction

 The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods

 Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was predetermined and was 1.5 to 5000mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. An additional dose level was selected in Experiment 2 in order to achieve both four non-toxic dose levels due to the change in methodology.

 

A Confirmatory Experiment was also performed in TA1535 (with and without metabolic activation) using the pre-incubation and plate incorporation methodology following the result from Experiment 2. The dose range was the same as Experiment 2 (15 to 5000 µg/plate).

 

 Results

 The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No toxicity was observed in the Confirmatory Experiment at any dose level, either in the presence or absence of metabolicactivation (S9-mix).

A greasy test item precipitate was noted at and above 1500mg/plate, this observation did not prevent the scoring of revertant colonies.


The test item induced statistically significant and reproducible increases in the frequency of TA1535 revertant colonies both with and without metabolic activation (S9-mix) in Experiment 2 and the Confirmatory Experiment (pre-incubation method). Statistically significant increases were also noted in the Confirmatory Experiment employing the plate incorporation method.

No significant increases in the frequency of revertant colonies were recorded in Experiment 1 or any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.

 

Conclusion

Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be mutagenic under the conditions of this test only inS. typhimuriumstrain TA1535, this response was reproducible when utilizing the pre-incubation method.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 28 September 2017 Experimental completion date 06 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
EC Number: 500-215-4
Physical state/Appearance: Clear yellow liquid
Batch: 52611021
Purity: 100% (UVCB*)
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Epoxy liquid
No correction for purity was made.
Target gene:
The purpose of the study was to assess the potential chromosomal mutagenicity of the test item, Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin, on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.
Species / strain / cell type:
primary culture, other: whole blood
Details on mammalian cell type (if applicable):
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 27 years
Main Experiment: male, aged 24 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Cytokinesis block (if used):
demecolcine (Colcemid 0.1 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (S9)
Test concentrations with justification for top dose:
Preliminary Toxicity Test
The dose range of test item used was 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.

Main Experiment
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2.5, 5, 10, 20, 40, 80 and 120 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 80, 160 and 240 µg/mL.
Vehicle / solvent:
The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL and the purity of the test item was 100%.
The test item was immiscible in aqueous media at 50 mg/mL and dimethyl sulfoxide at 250 and 500 µg/mL. However, the test item was miscible in acetone at 500 mg/mL in solubility checks performed in house. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration used in the Preliminary Toxicity Test was 2500 µg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.4 µg/mL for 4-hour exposure, 0.1 µg/mL for 24-hour exposure
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
4 µg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9 mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous, the cultures were established at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The Preliminary Toxicity Test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2.5, 5, 10, 20, 40, 80 and 120 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 5, 10, 20, 40, 80, 160 and 240 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed except for 24 hours, without S9-mix (MMC 0.1 µg/mL, B culture) as only 43 cells were analyzed due to excessive toxicity of the positive control compound. Of the 43 cells scored, only 8 of the cells were aberrant. The SD is confident that the study can be considered valid because there was a demonstrable increase in the frequency of cells with aberrations with the positive control in both cultures, thereby confirming the test system sensitivity
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
primary culture, other: whole blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 9.77 to 2500 µg/mL. The maximum dose was the maximum practical dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 39.06 µg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9), at and above 78.13 µg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 156.25 µg/mL in the 24-hour continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to the maximum dose level tested (2500 µg/mL) in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 120 µ/mL for the 4(20)-hour exposure group in the absence of S9, 160 µg/mL in the 4(20)-hour exposure group in the presence of S9 and was 240 µg/mL for the continuous exposure group.

Chromosome Aberration Test – Main Experiment
The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test with the exception that there was some evidence of toxicity in the upper dose levels of the 24-hour exposure group only which was not observed in the previous test. However, there were metaphases suitable for scoring present up to the maximum dose level of test item in all three exposure groups.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 80 µg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9), at and above 40 µg/mL in the 4(20)-hour exposure group in the presence of S9 and the 24-hour continuous exposure group.
They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed except for the 24-hour continuous exposure group.
In the 4(20)-hour exposure group in the absence of S9, the maximum dose level selected for metaphase analysis was 80 µg/mL, the lowest precipitating dose level.
In the presence of S9, the maximum dose level selected for metaphase analysis was 40 µg/mL, the lowest precipitating dose level.
In the 24-hour continuous exposure group, 24%, 57% and 66% mitotic inhibition was achieved at 10, 20 and 40 µg/mL, respectively. Above this dose level, the metaphases present were few in number. Therefore, the maximum dose level selected for metaphase analysis was 20 µg/mL, because this dose level achieved optimum toxicity as defined by the OECD test Guideline (55±5%).

The assay was considered valid as it met all of the following criteria:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range
• All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration ofFatty acids, C18-unsatd., dimers, polymers with epichlorohydrin(µg/mL)

4(20)-hour without S9

0*, 2.5, 5, 10, 20*, 40*, 80*, 120, MMC0.4*

4(20)-hour with S9 (2%)

0*, 2.5, 5, 10*, 20*, 40*, 80, 160, CP4*

24-hour without S9

0*, 5*, 10*, 20*, 40*, 80, 160, 240, MMC0.1*


*  = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP  = Cyclophosphamide

Conclusions:
Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

 Introduction

This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1991). 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Experiment were as follows:

Group

Final concentration of test itemFattyacids, C18-unsatd., dimers, polymers with epichlorohydrin(µg/mL)

4(20)-hour without S9

0, 2.5, 5, 10, 20, 40, 80, 120

4(20)-hour with S9 (2%)

0, 2.5, 5, 10, 20, 40, 80, 160

24-hour without S9

0, 5, 10, 20, 40, 80, 160, 240

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test item, Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 09 January 2018 Experimental completion date: 30 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
EC Number: 500-215-4
Physical state/Appearance: Clear yellow liquid
Batch: 52611021
Purity: 100% (UVCB*)
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Epoxy liquid
No correction for purity was made.
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.
Metabolic activation:
with and without
Metabolic activation system:
2% S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.63, 1.25, 2.5, 5, 10, 20, 40, 60, 80 µg/ml
These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test.

Mutagenicity Test
4-hour without S9 1.25, 2.5, 5, 10, 20, 40, 60, 80 µg/ml
4-hour with S9 (2%) 0.63, 1.25, 2.5, 5, 10, 20, 40, 60 µg/ml
24-hour without S9 1.25, 2.5, 5, 10, 20, 40, 60, 80 µg/ml
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments


Vehicle / solvent:
The test item was immiscible in aqueous media at 50 mg/mL and dimethyl sulfoxide at 250 and 500 mg/mL. However, the test item was miscible in acetone at 500 mg/mL in solubility checks performed in house. Therefore acetone was used as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
400 µg/mL and 150 µg/mL, respectively, in the 4-hour and 24-hour exposure groups
Positive control substance:
ethylmethanesulphonate
Remarks:
absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
presence of metabolic activation
Details on test system and experimental conditions:
Experimental Design and Study Conduct
Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test Procedure
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 0.63 to 80 µg/mL for all three of the exposure groups. These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item, vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Assessments
Measurement of Survival, Viability and Mutant Frequency
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
There was evidence of slight reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 24-hour exposure group when compared to the concurrent vehicle control group. Both in the 4-hour and 24-hour exposures in the absence of metabolic activation, a precipitate of the test item was observed at and above 60 µg/mL. In the 4-hour exposure in the presence of metabolic activation a precipitate was observed at and above 40 µg/mL. Therefore, the dose levels selected for the main test were based on precipitate.

Mutagenicity Test
There was no evidence of marked dose related toxicity following exposure to the test item in all of the three exposure groups, as indicated by the %RSG and RTG values. There was no evidence of any marked reductions in viability (%V) in either of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances. The concentration of 80 µg/mL in the 4-hour and 24-hour –S9 exposure groups, was not plated out for 5-TFT resistance and viability due to excessive precipitate. Precipitate of the test item was observed at 60 and 80 µg/mL in the 4-hour and 24-hour –S9 exposure groups. Precipitate of the test item was observed at 60 µg/mL in the 4-hour +S9 exposure group. The presence of one analyzable precipitating dose level in in all three exposure groups satisfies the requirements of the OECD 490 guideline.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, in any of the three exposure groups.


Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 0.63 to 80 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

0.63

96

87

90

1.25

91

95

104

2.5

87

103

112

5

85

100

98

10

75

86

107

20

78

95

85

40

80

96 p

105

60

93 p

92 p

78 p

80

80 p

87 p

62 p

p = precipitate at the end of exposure

Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it was considered to be non-mutagenic in this assay.
Executive summary:

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (Acetone), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group

Concentration of Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin (µg/mL)

 plated for viability and mutant frequency

4-hour without S9

2.5, 5, 10, 20, 40, 60

4-hour with S9 (2%)

24-hour without S9

Results……..

The maximum dose level used in the Mutagenicity Test was limited by precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose related (linear-trend) increases in either of the three exposure groups. The GEF value was not exceeded at any test item concentration.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it was considered to be non-mutagenic in this assay.

Additional information

Justification for classification or non-classification